Palatinose (isomaltulose) and trehalulose are produced on a large scale from sucrose by an enzymatic rearrangement reaction using immobilized bacterial cells. The α1->β2-glycosidic bond, which exists between the monosaccharides of the disaccharide sucrose, is isomerized, in the case of palatinose to an α1->α6 bond or, in the case of trehalulose an α1->α1 bond. This rearrangement of sucrose to give the two noncariogenic disaccharides is catalyzed by the bacterial enzyme sucrose isomerase, also referred to as sucrose mutase. Suitable sequences are described for example in WO 95/20047 (U.S. Pat. No. 5,786,140; U.S. Pat. No. 5,985,622).
Also described are sucrose isomerases from Erwinia rhapontici (palI gene, GenBank Acc. No.: AF279281; Börnke et al. (2001) J Bacteriol 183(8):2425-2430) and Klebsiella sp. Strain LX3 (GenBank Acc. No.: AY040843; Zhang et al. (2002) Appl Environ Microbiol (68):2676-2682).
WO 01/59136 describes methods for the direct production of noncariogenic sugars directly in transgenic plants which comprise recombinant nucleic acid molecules encoding proteins with the enzymatic activity of a sucrose isomerase. Described are expression constructs for said sucrose isomerase for expression in plants, and the transgenic plants transformed therewith.
WO 01/59135 describes methods for influencing the pollen development using anther-, tapetum- or pollen-specifically expressed sucrose isomerases. However, the constitutive expression of sucrose isomerase in plants has an adverse effect on plant growth (Börnke F et al. (2002) Planta 214:356-364).
The aim of plant biotechnology work is the generation of plants with advantageous novel properties, for example for increasing agricultural productivity, increasing the quality in the case of foodstuffs, or for producing specific chemicals or pharmaceuticals. The plants' natural defense mechanisms against pathogens are frequently insufficient. Fungal-diseases alone result in annual yield losses of many billions of US$. The introduction of foreign genes from plants, animals or microbial sources can increase the defenses. Examples are the protection of tobacco against feeding damage by insects by expressing Bacillus thuringiensis endotoxins under the control of the 35S CaMV promoter (Vaeck et al. (1987) Nature 328:33-37) or the protection of tobacco against fungal infection by expressing a bean chitinase under the control of the CaMV promoter (Broglie et al. (1991) Science 254:1194-1197). However, most of the approaches described only offer resistance to a single pathogen or a narrow spectrum of pathogens.
Only a few approaches exist which impart a resistance to a broader spectrum of pathogens, in particular fungal pathogens, to plants. Systemic acquired resistance (SAR)—a defense mechanism in a variety of plant/pathogen interactions—can be conferred by the application of endogenous messenger substances such as jasmonic acid (JA) or salicylic acid (SA) (Ward, et al. (1991) Plant Cell 3:1085-1094; Uknes, et al. (1992) Plant Cell 4(6):645-656). Similar effects can also be achieved by synthetic compounds such as 2,6-dichloroisonicotinic acid (INA) or S-methyl benzo(1,2,3)thiadiazole-7-thiocarboxylate (BTH; BION) (Friedrich et al. (1996) Plant J 10(1):61-70; Lawton et al. (1996) Plant J. 10:71-82). The expression of pathogenesis-related (PR) proteins, which are upregulated in the case of SAR, may also cause pathogen resistance in some cases.
In barley, the Mlo locus has been described for some time as a negative regulator of plant defense. The loss, or loss of function, of the Mlo gene causes an increased and, above all, race-unspecific resistance for example against a large number of mildew species (Büschges R et al. (1997) Cell 88:695-705; Jorgensen J H (1977) Euphytica 26:55-62; Lyngkjaer M F et al. (1995) Plant Pathol 44:786-790). The Mlo gene has only recently been cloned (Buschges R et al. (1997) Cell 88:695-705; WO 98/04586; Schulze-Lefert P, Vogel J (2000) Trends Plant Sci. 5:343-348). Various methods for obtaining pathogen resistance using these genes have been described (WO 98/04586; WO 00/01722; WO 99/47552). It is unclear whether an Mlo-based approach can also be performed successfully in dicotyledonous plants.
Phytopathogenic fungal species generally live as saprophytes or parasites. The latter depend—at least during certain phases of their lifecycle—on a supply of active substances (for example a supply of vitamins, carbohydrates and the like), as it can only be provided in this form by live plant cells. The expert classifies parasitic fungi as necrotrophic, hemibiotrophic and biotrophic. In the case of necrotrophic fungal parasites, the infection results in destruction of the tissue and thus in the death of the plant. In most cases, these fungi are only facultative parasites; they are just as capable of saprophytic multiplication in dead or dying plant material.
Biotrophic fungal parasites are characterized in that parasite and host cohabit, at least over prolonged periods. While the fungus withdraws nutrients from the host, it does not kill it. Most biotrophic fungi are obligate parasites. Hemibiotrophic fungi live temporarily as biotrophs and kill the host at a later point in time, i.e. they enter a necrotrophic phase.
A further, large group of biotrophic plant pathogens of enormous agro-economical importance are nematodes. Phytopathogenic nematodes feed on the outermost parts of plant tissue (ectoparasites) or, after penetration into the plant, in cell layers further in (endoparasites). Two groups of endoparasitic root nematodes are distinguished according to their lifestyle and nutrition: cyst nematodes (Heterodera and Globodera species) and root-knot nematodes (Meloidogyne species). Both groups are obligate biotrophic parasites which induce the development of specific feeder cells in the roots. These feeder cells are plant cells whose metabolism has been modified by the nematodes in such a way that they specifically serve the supply of nutrients to the developing nematodes. The development of endoparasitic root nematodes depends totally on these feeder cells (for a review, see Sijmons et al. (1994) Ann. Rev. Phytopathol. 32: 235-259). Cyst nematodes (Heterodera and Globodera species) remain at the parasitization site in the root (sessile endoparasites) and convert the surrounding cells by protoplast fusion into syncytia while dissolving some of the cell walls. The nematodes feed off these feeder cells, which are formed in the root's central cylinder, during which process the nematodes swell greatly in size. Root-knot nematodes (Meloidogyne species) likewise remain at the parasitization site, once chosen, and bring about the development of feeder cells which, in contrast to the cyst nematodes, consist of several, multinucleate giant cells which develop as a result of synchronous divisions of the nucleus without cell wall formation (Fenoll and Del Campo (1998) Physiol. Mol. Biol. Plants 4:9-18). The development of the feeder cell systems is induced by signal molecules in the nematodes' saliva. It is known that a series of plant genes change their expression profile greatly during these differentiation, processes. Promoters which are induced specifically in the feeder cell system (syncytia) are described in the literature. Those which may be mentioned by way of example are the tobacco Δ0.3 TobRB7 promoter (Opperman et al. (1994) Science 263:221-223), the tomato Lemmi9 promoter (Ecobar et al. (1999) Mol Plant Microbe Interact 12: 440-449) and the geminivirus V sense promoters (WO 00/01832).
WO 94/10320 describes DNA constructs for the expression of genes which act as inhibitors of endogenous plant genes (for example ATP synthase, cytochrome C, pyruvate kinase) under the control of nematode-induced promoters in den syncytia.
Despite several advances in some fields of plant biotechnology, success in achieving a pathogen resistance in plants has been very limited and as yet only sufficiently documented in the case of viruses. Yield losses in particular as a result of fungal and nematode attack are a serious problem; then as now, they require an intensive application of fungicides and nematicides. However, the problems which this entails have still not been tackled sufficiently.